Treating cancer using electromagnetic fields in combination with other treatment regimens

ABSTRACT

Chemotherapeutic treatment for certain cancers may be combined with low intensity, intermediate frequency alternating electric fields that are tuned to a particular type of target cell. When the tuned fields were combined with Paclitaxel, Doxorubicin or Cyclophosphamide, excellent results were obtained against human breast cancer cells (MDA-MB-231) and non-small cell lung (H1299) carcinomas in culture. More specifically, cell proliferation inhibition similar to that obtained by drug alone was reached by exposure to the combined treatment at drug concentrations between one and two orders of magnitude lower than for drug-only regimens of treatment.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of Ser. No. 11/695,176, filed Apr. 2,2007, which claims the benefit of U.S. provisional application60/744,295, filed Apr. 5, 2006, each of which is incorporated herein byreference.

BACKGROUND

As described in U.S. Pat. Nos. 6,868,289 and 7,016,725 each of which isincorporated herein by reference, and in U.S. patent application Ser.No. 11/111,439 (filed Apr. 21, 2005 and published as US2005/0209642) andSer. No. 11/537,026 (filed Sep. 29 2006), each of which is incorporatedherein by reference, intermediate frequency (100-300 kHz) alternatingelectric fields, (referred to herein as “TTFields”) damage as well asinhibit the growth of numerous types of cancer cells in vitro as well asa number of malignancies in vivo. The efficacy of the treatment isenhanced by sequentially applying fields of varying directions and bythe use of special insulated electrodes.

TTFields act by two mechanisms of action: First, they disrupt the normalpolymerization-depolymerization process of the spindle microtubulesduring mitosis. Secondly, they cause a physical disruption of cellstowards the end of cytokinesis by producing a unidirectional force onall charge, polar and polarizable intracellular constituents, pushingthem towards the narrow neck between the two daughter cells. See Kirson,E. D., et al., Disruption of cancer cell replication by alternatingelectric fields, Cancer Res., 2004. 64(9): p. 3288-95, which isincorporated herein by reference.

Drugs and radiation therapy are more conventional approaches to treatingcancer. One example is Cisplatin or cis-diamminedichloroplatinum(II)(CDDP), which is a platinum-based chemotherapy drug used to treatvarious types of cancers, including sarcomas, some carcinomas (e.g.small cell lung cancer and ovarian cancer), lymphomas and germ celltumors. It was the first member of its class, which now also includescarboplatin and oxaliplatin. Another example is Paclitaxel, morecommonly referred to by the trade name Taxol®, which is a member of thelarger family of compounds known as taxanes. Currently, Paclitaxel isused in the treatment of breast, ovarian, certain non-small-cell lungcancers, and Kaposi's sarcoma.

SUMMARY OF THE INVENTION

Chemotherapeutic treatment for certain cancers are combined with lowintensity, intermediate frequency alternating electric fields that aretuned to a particular type of target cell to inhibit the growth of thecancer cells. In many cases, the resulting cell proliferation inhibitionis significantly higher than the inhibition obtained by drug-onlyregimens of treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts an electrodes arrangement for applying TTFields to an invitro specimen.

FIGS. 2A and 2B depict the results of cytotoxicity calibrationexperiments for Cisplatin and Taxol, respectively, on MDA-231 cells.

FIGS. 3A and 3B depict the results of cytotoxicity calibrationexperiments for Cisplatin and Taxol, respectively, on B16F10 cells.

FIG. 4 depicts the cell proliferation measured in an experiment on humanbreast cancer (MDA-231) cells.

FIG. 5 depicts the cell proliferation measured in an experiment on mousemelanoma (B16F10) cells.

FIG. 6 depicts the recovery of the rate of cell proliferation 24 hoursafter treatment was stopped.

FIG. 7 depicts experimental results that show the frequency dependenceof the TTFields anti-proliferation efficacy for human breast cancer(MDA-231) cells.

FIG. 8A depicts the measured relationship between TTFields intensity andcell proliferation rate for different field intensities for MDA-231cells.

FIG. 8B depicts the dose-response curve for MDA-231 cell line subjectedto increasing concentrations of Paclitaxel, alone and in combinationwith TTFields of different intensities.

FIG. 8C depicts the dose-response curve for MDA-231 cell line subjectedto increasing concentrations of Doxorubicin, alone and in combinationwith TTFields of different intensities.

FIG. 8D depicts the dose-response curve for MDA-231 cell line subjectedto increasing concentrations of Cyclophosphamide, alone and incombination with TTFields of different intensities.

FIGS. 9A, 9B and 9C depict the results of experiments on ER-negativeMDA-231 cells exposed to TTFields and three different chemotherapeuticagents for different durations.

FIG. 9D illustrates the impact of how long the TTFields are applied whenTTFields are used alone and in combination with Cyclophosphamide.

FIG. 10A depicts the relationship between the intensity of the 200 kHz

TTFields (applied alone) and the cell proliferation rate for non-smallcell lung carcinoma.

FIG. 10B depicts the dose-response curve for H1299 cell line, subjectedto increasing concentrations of Paclitaxel, and in combination withTTFields.

FIG. 11A is an Isobolographic plot for Paclitaxel at differentconcentrations and TTFields at different intensities on MDA-231 cells.

FIG. 11B is an Isobolographic plot for Paclitaxel at differentconcentrations and TTFields at different intensities on H1299 cells.

FIG. 11C is an Isobolographic plot for Doxorubicin at differentconcentrations and TTFields at different intensities on MDA-231 cells.

FIG. 11D is an Isobolographic plot for Cyclophosphamide at differentconcentrations and TTFields at different intensities on MDA-231 cells.

FIG. 12 is a schematic block diagram of an apparatus for applying anelectric according to one exemplary embodiment for selectivelydestroying cells.

FIG. 13 is a simplified schematic diagram of an equivalent electriccircuit of insulated electrodes of the apparatus of FIG. 12.

FIG. 14 is a cross-sectional illustration of a skin patch incorporatingthe apparatus of FIG. 5 and for placement on a skin surface for treatinga tumor or the like.

FIG. 15 is a cross-sectional illustration of the insulated electrodesimplanted within the body for treating a tumor or the like.

FIGS. 16A-16D are cross-sectional illustrations of various constructionsof the insulated electrodes of the apparatus of FIG. 12.

FIG. 17 is a front elevational view in partial cross-section of twoinsulated electrodes being arranged about a human torso for treatment ofa tumor container within the body, e.g., a tumor associated with lungcancer.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

When used as the only treatment modality, the therapeutic efficacy ofTTFields was found to be high and the therapeutic index extremely high(few or no side effects), however, treatment duration was relativelylong and the required field intensities were relatively high. In orderto improve the treatment efficacy, the effects of combining TTFieldswith other treatment modalities was tested. It was hypothesized thatsuch a combination would be beneficial regardless of whether themechanism of action of the two (or more) modalities was similar ordifferent, and experiments were conducted to test this hypothesis. Theresults of those experiments are described below. In each of theexperiments, a TTField treatment protocol previously shown to beeffective was selected, and the efficacy of the TTFields and each agentalone were compared to the efficacy of the combined treatment withTTFields and each of the agents.

First Set of Experiments

In a first set of experiments, TTFields were applied (with the fielddirection alternating between two directions) to human breast cancer(MDA231) and mouse melanoma (B16F10) cells in culture, both with andwithout a chemotherapeutic agent. Taxol and Cisplatin were selected asthe agents because they have different mechanisms of action.

The MDA-231 and B16F10 cells were obtained from ATCC (USA). Both typesof cells were cultured in DMEM +10% FCS media (Biological IndustriesLtd., Israel) in CO₂ incubator (5% CO₂) at 37° C. Cell resuscitation wasdone using Trypsin/EDTA solution (0.25%/0.02%, Biological IndustriesLtd., Israel). The experiments were performed in 35 mm Petri dishes(NUNC, USA). Cisplatin and Taxol were obtained from Sigma (USA). A cellproliferation assay kit was obtained from Biological Industries Ltd.,Israel.

Cells, grown in 25 cm² cell culture flasks, were removed usingTrypsin/EDTA solution (0.25%/0.02%), diluted with complete media tofinal concentration of 75×10³ cells per ml. 200 μl of diluted suspensionwere placed as a drop in the centre of 35 mm Petri dish and incubatedfor 24 hours. The initial cell number was measured as a light absorptionby formazan produced by cells during 2 hours using the XTT method andexpressed as OD₀. (XTT is sodium3′-[1-(phenyl-amino-carbonyl)-3,4-tetrazolium]-bis(4-mrthoxy-6-nitro)-benzenesolfonic acid hydrate. XTT is cleaved to formazan (absorbs at 450-500nm) by the “succinate-tetrazolium reductase” system of the respiratorychain of the mitochondria and is active only in viable cells. Therefore,the amount of formazan dye (A_(450 nm)) formed directly correlates tothe number of metabolically active cells in culture. The XTT assay iswidely used for the measurement of cell proliferation in response togrowth factors, cytokines, mitogens, nutrients, anti-cancer drugs andphysiological mediators.)

The media in the Petri dish was replaced by fresh media (3 ml with orwithout Taxol or Cisplatin), thermo-couples were placed at the center,and the dish cover was replaced by one with attached electrodes. Cellsamples without TTField treatment were placed in CO₂ incubator at 37° C.for 24 hours, while TTField treated samples were placed in CO₂ incubatorat 25° C., also for 24 hours. Final incubation temperature of TTFieldtreated samples was 37±0.7° C. due to heating induced by TTFields (asmeasured by inserted thermo-couples). At the end of 24 hour treatment,the final cell number was measured as an absorption by formazan producedby cells during 2 hours using XTT method and expressed as OD₁.

The rate of cell proliferation was calculated as a ratio of the finalcell number to the initial cell number (OD₁/OD₀). An OD₁/OD₀ ratio of 1means that the no increase in cell number, i.e., a complete cellproliferation arrest is achieved. The change in the cell numberproliferation rate was calculated as(OD₁/OD₀−1)_(EXPERIMENT)/(OD₁/OD₀−1)_(CONTROL).

In order to evaluate the ability of treated cells to recover, the cellswere incubated in normal media after treatment removal for additional 24hours, and the number of cells was measured as an absorption by formazanproduced by cells during 2 hours using XTT method and expressed as OD₂.The rate of cell proliferation OD₂/OD₁ was calculated as a ratio offinal cell number (after the additional incubation period) OD₂ perinitial cell number (before the additional incubation period) OD₁.

For those samples that were subjected to TTFields, two-directional 200kHz sinusoidal TTFields were generated by an appropriate waveformgenerator and an amplifier. The output of the amplifier was switchedbetween two pairs of outputs every 250 mSec, with the outputs connectedto two pairs of electrodes, insulated by a high dielectric constantceramic (e.g., PMN-PT, EDO Corporation, Utah), positioned in the Petridish as depicted in FIG. 1. Field intensity in the medium surroundingthe cells was measured to be approximately 7 V/cm. Thus, each pair ofparallel electrodes was activated at a duty-cycle of 50% (250 mSecON-250 mSec OFF) such that when one pair was ON, the other pair was OFF.

Before the main experiments were conducted, calibration experiments wereperformed to determine the doses of Cisplatin and Taxol that should beused in the main experiments for the two representative cell culturesstudied. The purpose of these calibration experiments was to find thedosage of the respective drug that, taken alone (i.e., withoutTTFields), provided a cytotoxicity such that about 50% of the cells arekilled within the 24 hour study period. FIGS. 2A and 2B depict theresults of these calibration experiments for Cisplatin and Taxol,respectively, on MDA-231 cells; and FIGS. 3A and 3B depict the resultsof these calibration experiments for Cisplatin and Taxol, respectively,on B16F10 cells. On the basis of the calibration experiment, a drugconcentration of 15 μM was chosen for the main experiment with Cisplatinand, and a drug concentration of 0.05 μM was chosen for the mainexperiment with Taxol.

After the drug concentrations were selected (based on the calibrationexperiments), the main experiments were performed to determine theeffects of (a) each drug taken alone; (b) two-directional TTFields takenalone; and (c) both drugs and two-directional TTFields.

FIG. 4 depicts the results of the main experiment on human breast cancer(MDA-231) cell proliferation, as measured by the XTT assay. It can beseen see that Taxol (0.05 μM) and Cisplatin (15 μM) alone reduced cellproliferation by 70% and 63% respectively as compared with the control.The TTFields alone (labeled “exp.”) reduced cell proliferation by 89%.The combination of TTFields with Taxol (labeled “exp. with Taxol”) orCisplatin (labeled “exp. with Cisplatin”) led to an increase inproliferation arrest. In the case of Cisplatin there was complete cellproliferation arrest OD₁≈OD₀, and when Taxol was used in combinationwith TTFields there was an absolute reduction of the number of cellsindicating that on top of complete proliferation arrest (about 40% ofthe cells died under the influence of the combined treatment).

FIG. 5 depicts the results of the main experiment on mouse melanoma(B16F10) cell proliferation, as measured by the XTT assay. It can beseen that Taxol (0.05 μM) (labeled “control with Taxol”) and Cisplatin(15 μM) (labeled “control with Cisplatin”) alone reduced cellproliferation to a lesser extent than the combined effect of either drugin combination with the TTFields (labeled “exp. with Taxol” and “exp.with Cisplatin”, respectively).

FIG. 6 depicts the recovery of the rate of cell proliferation that wasobserved 24 hours after treatment removal, which can serve as anadditional index of the treatment potency. FIG. 6 contains four pairs ofbars. Within each pair, the left bar represents OD₁/OD₀, and the rightbar represents OD₂/OD₁. The results demonstrate that there is completerecovery of proliferation after Taxol removal (see the large OD₂/OD₁ barlabeled “control with Taxol”). In marked contrast, there is no cellrecovery after either TTFields treatment alone or after combined Taxoland TTField treatment (see the small OD₂/OD₁ bars labeled “experimentw/o Taxol” and “experiment with Taxol”).

Second Set of Experiments

In a second set of experiments, TTFields were applied (with the fielddirection alternating between two directions) to human breast cancer(MDA-MB-231) and non-small cell lung carcinoma (H1299) cells in culture,both with and without each of three chemotherapeutic agents (Paclitaxel,Doxorubicin and Cyclophosphamide) in various concentrations

Human breast cancer (MDA-MB-231) and human non-small cell lung cancer(H1299) cells were obtained from ATCC (USA). The cells were cultured inDMEM +10% FCS media (Biological Industries Ltd., Israel) in a 5% CO₂incubator at 37° C. The chemotherapeutic agents: Taxol (Paclitaxel),adriamycin (Doxorubicin) and Cyclophosphamide were obtained from Sigma,USA. The stock solution of Paclitaxel was prepared in DMSO (Sigma USA)at concentration of 5 mM. The stock solutions of Doxorubicin andCyclophosphamide were prepared in phosphate buffered saline atconcentrations of 8.5 mM and 3.0 M respectively. All stock solutionswere stored at −20° C. and were freshly diluted with media shortlybefore their introduction to the cultured cells.

In the experimental set up, cells grown in 25 cm² cell culture flaskswere removed using trypsin/EDTA (0.25%/0.02%) solution (BiologicalIndustries Ltd., Israel), diluted with the media described above, to afinal concentration of 100×10³ cells per ml. 200 μl of dilutedsuspension were placed as a drop at the centre of 35 mm Petri dishes(NUNC, USA). After the dishes were incubated for 2 hours at 37° C., toallow for cell attachment, 1.5 ml of complete media was added and cellswere incubated for an additional 22 hours (pre-incubation).

After pre-incubation, the initial cell number was estimated usingstandard XTT method (Cell proliferation assay Kit, Biological IndustriesLtd., Israel) by measuring the light absorption by formazan, produced bycells during a period of 2 hours, and expressed as OD₀. The media in thePetri dishes was then replaced by fresh media (3 ml), with or without achemotherapeutic agent. Temperature was continuously measured by athermocouple (Omega, UK) placed at the center of the dish. Asillustrated in FIG. 1, two pairs of electrodes 110, 120, insulated by ahigh dielectric constant ceramic (PMN-PT, EDO Corporation, Utah),connected to sinusoidal waveform generator—TTField generator (NovoCureLtd., Haifa, Israel), were positioned in all Petri dishes 130 (includingcontrols) so as to alternately generate electric fields in two differentdirections around the cultured cells 140.

Control cell dishes that did not receive TTFields treatment, were placedin a CO₂ incubator at 37° C. for 24 hours while TTFields treated disheswere placed in a CO₂ incubator in which temperature was controlled suchthat the final incubation temperature of treated dishes was 37±0.5° C.At the end of 24 hours treatment, the cell number was estimated againusing the XTT method and expressed as OD₁ (the light absorption byformazan produced by cells during a period of 2 hours). The rate of cellproliferation was expressed as the OD₁/OD₀ ratio. The OD₁/OD₀ ratio foruntreated cells was in the range of 2.0±0.2 for both cell lines studied,i.e. the cell number doubled during the 24 hour incubation. Treatmentefficacy is expressed as the change in the rate of cell proliferation,presented as a % of control, calculated for each experiment by thefollowing equation:

(OD ₁ /OD ₀)_(EXPERIMENT)*100%/(OD ₁ /OD ₀)_(CONTROL).

To optimize the field effect, two fields of perpendicular direction weregenerated sequentially in an alternating pattern by switching the outputof the amplifier between the two pairs of electrodes every 250 ms. Theelectric field intensity in the culture medium was measured using aprobe consisting of two 0.25 mm diameter insulated wires with exposedtips 1 mm apart, which was dipped in the culture media at the centre ofthe Petri dish. A high-input impedance differential amplifier translatedthe alternating potential difference amplitude into a corresponding DCvoltage that was recorded. Note that field intensities used throughoutthis specification are expressed in peak voltage amplitude differenceper centimeter distance (V/cm).

Four different runs were conducted in conjunction with eachchemotherapeutic agent: untreated control, treatment with eitherTTFields alone, treatment with one of the chemotherapeutic agents alone,and combined TTField—chemo treatment.

The Chou and Talley method for assessing the combined effect of multipledrugs was used for the drug—TTFields combinations. TTField intensityreplaced the classical concentration variable in the analyses.Dose-response curves were generated for TTFields and each drugseparately to determine the median effect plots. Variable ratios of drugconcentrations—TTFields intensities were used to determine theCombination indexes (Cist):

CI=(C _(A,x) /Ix _(x,A))+(B _(B,x) /IC _(x,B))

Where: C_(A,x) and C_(B,x) are the concentrations (intensities) oftreatment A and treatment B used in combination to achieve apredetermined x % effect. IC_(x,A) and IC_(x,B) are the correspondingconcentrations (intensities) for any single agent to achieve the sameeffect. In all cases herein, A represents TTFields and B represents thechemotherapeutic drug.

This analysis allows drugs with different mechanisms of action to beassessed. A CI<1 denotes synergy (more than additive), a CI of 1reflects summation (additive), and a CI>1 indicates antagonism (lessthan additive).

Isobologram analyses, for evaluation of the nature of interaction of twoagents, were performed as follows: The concentrations (intensities) ofagent A and B required to produce a defined single-agent effect of, forexample, IC₅₀, IC_(x,A) and IC_(x,B), are plotted on the x and y axes ina two-coordinate plot, corresponding to (C_(A), 0) and (0, C_(B)),respectively. The line connecting these two points is the line ofadditivity. The concentrations (intensities) of the two agents used incombination to provide the same selected level of effect denoted aspoint (C_(A), C_(B)), are introduced on the same plot. Synergy,additivity, or antagonism are indicated when the point (C_(A), C_(B)) islocated below, on, or above the line, respectively.

Results for Human Breast Cancer (MDA-MB-231)

Since the sensitivity of different cell types to the TTFields frequencyis different, an initial round of experiments was run to determine whichfrequency is most effective for each type of target cell. FIG. 7 depictsthe results of those initial experiments, and shows the frequencydependence of the TTFields anti-proliferation efficacy for human breastcancer cells (MDA-MD-231). The data indicates a peak effectiveness at150 kHz. Note that in the initial experiments, the TTField intensity waskept constant at 1.75 V/cm at all frequencies. Each point representsmean values±SEM of 18-36 samples, and all effects were statisticallysignificant. In FIG. 7, * indicates a student's t test, P<0.01 relativeto the control, and ** indicates a student's t test, P<0.01 relative tothe experiments at 100 & 200 kHz.

A second round of experiments was then performed to determine theproliferation rate of ER-negative MDA-MB-231 cells (as % of control)after 24 hour exposure to Paclitaxel, Doxorubicin and Cyclophosphamidealone and in combination with TTFields at different intensities. FIGS.8A-8D depict the results of these experiments.

FIG. 8A depicts the measured relationship between TTFields intensity,and cell proliferation rate at field intensities of: 0.63, 1.25, 1.75and 2.95 V/cm, when the field was applied alone (i.e., without anydrugs) at the preselected frequency of 150 kHz. At the lowest fieldintensity of 0.63 V/cm there was no significant change in theproliferation rate (1.0±3.0%). At TTFields intensities of 1.25, 1.75 and2.95 V/cm there was a significant decrease in the cell proliferationrate: 10±3%, 26±4% and 75±5%, respectively. The TTFields intensityrequired for complete proliferation arrest, i.e. a 50% decrease in theproliferation rate, was calculated from the slope of the curve to be2.35 V/cm. In FIG. 8A, the symbols represent average values of 18samples obtained from three experiments, and the bars represent meanvalues±SEM.

Note that in FIGS. 8B-8D, data points with an open circle “∘” representthe drug alone; the squares “▪” represent the drug in combination withTTFields of 0.625 V/cm; the triangles “▴” represents the drug incombination with TTFields of 1.25 V/cm; and the closed circles “”represent the drug in combination with TTFields of 1.75 V/cm. Each pointrepresents mean values±SEM of 18 to 36 replicate measurements.

FIG. 8B depicts the dose-response curve for MDA-MB-231 cell linesubjected to increasing concentrations of Paclitaxel, in the range of0.01-500 nM, both alone and in combination with TTFields of differentintensities. A steep decrease in the cell proliferation rate is observedfor the drug-only treatment when Paclitaxel concentration increases from1.0 to 100 nM. At concentrations above 100 nM the proliferation ratestabilizes around the 50% level (e.g. complete arrest of cellproliferation without induction of cell death at this point in time).The dashed horizontal line represents a 60% of cell proliferation rate.(Note the inverse relationship between the rate of cell proliferationand the inhibitory effect of treatment, i.e., a 40% cell proliferationrate is equivalent to a 60% inhibition.)

It can be seen that low intensity TTFields (0.625 V/cm), in combinationwith Paclitaxel, have the same effect on cell proliferation rate asPaclitaxel alone at all Paclitaxel concentrations. In contrast, thecombination of Paclitaxel and TTFields of higher intensities (1.25 and1.75 V/cm) leads to a statistically significant (ANOVA, P<0.05)additional decrease in cell proliferation rate. The increase in cellgrowth inhibition by Paclitaxel, with and without TTFields, levels offat high Paclitaxel concentrations.

FIG. 8C depicts the dose-response curve for MDA-MB-231 cell linesubjected to increasing concentrations of Doxorubicin, both alone and incombination with TTFields of different intensities. For the drug-onlytreatment, it is apparent that cell proliferation rate decreases withincrease in Doxorubicin concentration until complete arrest (50%inhibition) is obtained at a concentration of about 1 μM. The dashedline represents 50% decrease in cell proliferation rate (i.e., 50%inhibition).

Once again, low intensity TTFields (0.625 V/cm) had no significanteffect on cell proliferation when applied both alone and in combinationwith all concentrations of Doxorubicin. The combination of TTFields ofhigher intensities (1.25 and 1.75 V/cm) with Doxorubicin results in astatistically significant (ANOVA, P<0.05) anti-proliferation effectwhich is added to the one obtained by the drug alone. This enhancedinhibition is observed throughout the Doxorubicin concentration rangeused in the experiments. The concentrations of Doxorubicin required toreach complete arrest of cell proliferation (50% inhibition) duringcombined treatment is 0.41 μM and 0.22 μM for 1.25 V/cm and 1.75 V/cmTTFields respectively.

FIG. 8D depicts the dose-response curve for MDA-MB-231 human cell linesubjected to increasing concentrations of Cyclophosphamide, alone, andin combination with TTFields of different intensities. In the absence ofTTFields, Cyclophosphamide concentrations of up to 10 mM produce nosignificant changes in proliferation rate. Higher concentrations resultin precipitous drop in the dose-response curve, and complete arrest ofcell proliferation (50% inhibition) is seen at 30.0 mM ofCyclophosphamide. (The dashed line represents 50% inhibition.) Higherconcentrations result in a further reduction of the number of cells.

The combined effect of Cyclophosphamide and TTFields has an additionalanti-proliferation effect which becomes apparent even at the lowestconcentrations used (statistically significant, ANOVA, P<0.05). Theconcentrations of Cyclophosphamide required to reach complete cellproliferation arrest (50% inhibition), during combined treatment are:15.2 mM, 10.0 mM and 6.2 mM for 0.63 V/cm, 1.25 V/cm and 1.75 V/cmTTFields intensities, respectively. These values compare to 30 mM forcomplete cell proliferation arrest with the drug alone.

The results depicted in FIGS. 8A-D are for the cell proliferation rateat the end of 24 hours of treatment. However, the induction of celldamage may take 3-4 days due to accumulation of the damaged structuresor molecules and the induction of cell suicidal pathway—apoptosis, andnecrosis. Therefore another set of experiments was performed to comparethe number of viable cells in culture along a period of 72 hours, whenone set of cells was treated continuously for the entire 72 hour periodby TTFields alone, drugs alone, or drugs in combination with TTFields,while the other was treated only for 24 hours and thereafter incubatedunder normal conditions for 48 hours.

FIG. 9A, 9B and 9C depict the results of these 72 hour experiments onER-negative MDA-MB-231 cells exposed to TTFields and three differentchemotherapeutic agents for different durations. In all three of thosefigures, the dashed line represents the untreated control, the opensymbols represent 24 hour treatment, and the filled symbols represent 72hour treatment, as summarized below in Table 1. Each point representsmean values±SEM of 24 replicate measurements obtained from 4experiments.

TABLE 1 ∘ Treatment with TTFields  Treatment with TTFields alone for 1day alone for 3 days □ Treatment with drug ▪ Treatment with drug alonefor 1 day alone for 3 days Δ combined treatment ▴ combined treatment for1 day for 3 days

FIG. 9A depicts the data for 12.5 nM Paclitaxel and 1.75 V/cm TTFields,both individually and combined, for both 24 and 72 hour treatmentregimens. It is apparent that when cells are treated with for 24 hourswith the TTFields alone (∘) or the Paclitaxel alone (□) a similarreduction in proliferation rate and the corresponding cell number isobtained at the end of treatment (approx. 27±3%, lower than control).Subsequently, complete recovery of cell proliferation rate is seen; withthe cell number approximately doubling every 24 hours of incubation. (A)24-72 hours treatment with TTFields and Paclitaxel. When treatment iscontinued for an additional period of 48 hours, for TTFields alone ()the cell number increases at a low rate (1.29 times per 24 h), while forPaclitaxel alone (▪) proliferation completely stops during second day oftreatment and the cell number is reduced during the third day. Combinedtreatment with TTFields and Paclitaxel for both 24 hours (Δ) and 72hours (▴) leads to induction of cell death already during the first 24hours, with cell count continuing to fall throughout the 72 hours periodeven in the case (Δ) when the treatment is no longer being appliedduring the last 48 hours.

FIG. 9B depicts the data for 0.1 μM Doxorubicin and 1.75 V/cm TTFields,both individually and combined, for both 24 and 72 hour treatmentregimens. It is apparent that treatment for 24 hours with theDoxorubicin alone (□) leads at the end of treatment to a reduction incell number by 26±4% as compared to the control. During the following 48hours, a slow increase in cell number is observed. Treatment for 72hours by TTFields alone () shows almost exactly the same cell countprofile. In contrast during a 72 hour long treatment with Doxorubicin(▪) there is a complete arrest of cell division at the end of the secondday and a small reduction in cell number after an additional 24 hours oftreatment. The combined treatment of cells with both TTFields andDoxorubicin (Δ) leads to complete halt of cell division after first 24hours. Induction of cell death is seen already at this point of time andcontinues during following 48 hours (even after the treatment is notbeing applied). A 72 hour long treatment period (▴) results in an effectthat is roughly similar to the 24 hour treatment (Δ).

FIG. 9C depicts the data for 20 mM Cyclophosphamide and 1.75 V/cmTTFields, both individually and combined, for both 24 and 72 hourtreatment regimens. This figure shows that when cells are treated withthe Cyclophosphamide alone for 24 hours (□) a reduction in cell numberis obtained (approx. 27±2%). Cessation of the treatment at this point(after 24 hours) leads to almost complete recovery of the cellproliferation such that their number approximately doubles every 24hours. Treatment by either Cyclophosphamide alone (▪) or TTFields alone() for a period of 72 hours results in a linear relatively slowincrease in the cell number. The combined treatment with both TTFieldsand Cyclophosphamide for either 24 hour (Δ) or 72 hour (▴) period leadsto a marked induction of cell death. After 24 hours the cell count is40±3% lower as compared to their number before treatment initiation. Atthe end of 72 hours there is almost complete loss of cells.

FIG. 9D illustrates the impact of how long the TTFields are applied when1.75 V/cm TTFields are used alone (open columns) and when 1.75 V/cmTTFields are used in combination with 30 mM Cyclophosphamide (solidcolumns). The data indicates that when TTFields are used alone, shortduration treatment of 12 hours or less is ineffective, but the 24 hourlong treatment is effective (as are treatments for longer durations, asevidenced by the filled circles “” in FIGS. 9A, 9B & 9C). In contrast,when the TTFields are combined with Cyclophosphamide (filled columns),the treatment is not effective when the TTFields are applied for 1 hour,but becomes fully effective when the TTFields are applied for 6 hours ormore. This behavior may indicate a specific interaction between the twoagents (see the discussion below). Note that in FIG. 9D, each columnrepresents mean values±SEM of 18 replicate measurements obtained from 3experiments. *P<0.01, student's t test relative to control. **P<0.01,student's t test relative to Cyclophosphamide alone.

Results for Non-Small Cell Lung Carcinoma (H1299)

For human non-small cell lung cancer (H1299). Initial testing indicatedthat the TTFields are most effective at a frequency of 200 kHz. As aresult, 200 kHz was selected for used in subsequent experiments tomeasure the effects of 24 hour exposure to Paclitaxel, Doxorubicin, andCyclophosphamide alone and in combination with TTFields at differentintensities.

FIG. 10A depicts the relationship between the intensity of the 200 kHzTTFields (applied alone) and the non-small cell lung carcinoma cellproliferation rate. It is seen that cell proliferation decreases asTTFields intensity is increased. The intensity of TTFields required forcomplete proliferation arrest (a 50% decrease in the rate of cellproliferation), as calculated from the slope of the curve, is 2.0 V/cm.In FIG. 10A, the symbols represent average values of 18 samples obtainedfrom three experiments and the bars represent±SEM.

FIG. 10B depicts the dose-response curve for H1299 cell line, subjectedto increasing concentrations of Paclitaxel alone (∘), and in combinationwith TTFields (, ▴), as a % of control. For Paclitaxel alone, a steepdecrease in cell proliferation rate is observed when Paclitaxelconcentrations increase from 1.0 to 200 nM. At concentrations above 200nM the proliferation rate reaches 55%, i.e. almost complete arrest ofcell proliferation. The combined effect of Paclitaxel and TTFields of1.25 V/cm (▴) and 1.75 V/cm () intensities leads to a significantadditional decrease in cell proliferation rate at all the concentrationsstudied. At the higher concentrations of Paclitaxel, when in combinationwith TTFields, cell death is induced. This effect that is not observedwhen Paclitaxel was used alone at concentrations up to 500 nM. In thisfigure, each point represents mean values±SEM of 24 to 32 replicatemeasurements, and the dashed line represents 60% of cell proliferationrate (i.e., 40% inhibition).

Discussion of Results

The experimental results demonstrate that in general TTFields haveeither additive or synergistic effects with Paclitaxel, Doxorubicin andCyclophosphamide for treatment of breast carcinoma and non-small cellcarcinoma of the lung. As these three drugs are known to have differentpharmacological mechanisms of action, it is not surprising that thedetails of the nature of their combined efficacy with the TTFieldsdiffer.

Paclitaxel is among the most commonly used microtubule-disrupting agentsfor the treatment of late-stage human breast cancer. Mechanistically, itexerts its anticancer actions primarily through disturbing thedisassembly of microtubules, consequently resulting in mitotic arrestand cell death. This mechanism is similar to that reported for TTFields.The observed results show that TTFields enhance the anti-proliferationeffect of Paclitaxel on both human breast cancer and non-small cell lungcancer cells, as explained above in connection with FIGS. 8 and 10.

The combination indexes (CI) obtained for human breast cancer(MDA-MB-231) and human non-small cell lung carcinoma (H1299) cellstreated with different drugs in combination with TTFields of variousintensities are summarized below in Table 2. It is seen that thecombined effects of Paclitaxel, like the other agents, vary fromadditivity to synergism as can be deduced from the fact that the valuesof all the calculated Combination Indexes in Table 2 are less than 1.

TABLE 2 Combination index TTFields MDA-MB-231 cells H1299 cellsintensity Paclitaxel Doxorubicin Cyclophosphamide Paclitaxel (V/cm) CI₄₀CI₅₀ CI₅₀ CI₄₀ 0.625 — — 0.74 — 1.25 0.97 0.99 0.84 0.73 1.75 0.86 0.980.95 0.98

Note that for Paclitaxel, the combination indexes for 60% proliferationrate equivalent to 40% inhibition level (CL₄₀) was used instead of themore commonly used CI₅₀, because the effects approach saturation abovethis level.

The combined effects of combination of TTFields of different intensitieswith chemotherapeutic agents of different concentrations can also beevaluated by means of isobolographic analysis. FIG. 11A is anIsobolographic plot for Paclitaxel at different concentrations andTTFields at different intensities on breast carcinoma MDA-MB-231; andFIG. 11B is an Isobolographic plot for Paclitaxel at differentconcentrations and TTFields at different intensities on non-small lungcarcinoma H1299 cells. Synergism at the low concentrations isdemonstrated by the fact that the data points fall below the isoboleline. In FIGS. 11A and 11B, the two points on the axes represent the 40%response levels for Paclitaxel alone and TTFields alone.

FIG. 11C is an Isobolographic plot for Doxorubicin at differentconcentrations and TTFields at different intensities on MDA-MB-231cells; and FIG. 11D is an Isobolographic plot for Cyclophosphamide atdifferent concentrations and TTFields at different intensities onMDA-MB-231 cells. In FIGS. 11C and 11D, the two points on the axesrepresent the 50% response levels for the respective drug alone andTTFields alone. Note that in all four of the Isobolographic plots (FIGS.11A-D), the solid line is the linear isobole, and the filled symbolsrepresent responses obtained with combinations.

At high concentrations of Paclitaxel and TTFields intensities, theinteraction in breast cancer approaches additivity. One may interpretthese findings as an indication that the two agents affect the tubulinand disrupt normal microtubule functions during mitosis in a similarway, but at two different sites or receptors. This conclusion iscompatible with the fact that proliferation inhibition by both highconcentrations of Paclitaxel (FIG. 8B), and the combined applicationasymptotically approach complete proliferation arrest. Similar mode ofinteraction and synergism was observed for Paclitaxel with antitubulinagent, 2-Methoxyestradiol (2-MeO-E₂) on MDA-MB-231 cells. Combinedtreatment with TTFields and Paclitaxel also shows synergistic mode ofinteraction for non-small lung carcinoma (H1299) cells, but at differentTTFields/Paclitaxel ratios as compared to MDA-MB-231 cells. The lungcells show lower sensitivity to Paclitaxel and higher sensitivity toTTFields when applied separately. In combinations, TTFields of lowerintensities with Paclitaxel of higher concentrations are required to getsynergism between these two treatment modalities.

Doxorubicin (Adriamycin) is an antibiotic that has a broad spectrum ofactivity both in experimental tumor models and in human malignancy.Table 2 indicates that the combined effect of Doxorubicin and TTFieldson MDA-MB-231 cells is additive (CI₅₀=1). This result is compatible withthe isobolographic analysis of the combination given in FIG. 11C.

The most pronounced synergism was found for theTTFields—Cyclophosphamide combination at the wide range ofconcentrations. The synergism of the Cyclophosphamide—TTFieldscombination on the inhibition of MDA-MB-231 cells proliferation isapparent from both Table 2 and FIG. 8D. As seen in FIG. 11D, thesynergism increases with Cyclophosphamide concentration, an oppositetrend when compared to the concentration dependence of theTTFields—Paclitaxel combination for these cells.

The observed results may be attributable to the mechanism of action ofTTFields. Of special significance is the fact that exposure to TTFieldsfor 12 hours or less has no effect on cell proliferation while similarexposure, in combination with Cyclophosphamide, significantly shortensthe minimal duration of exposure required to achieve a significanteffects. The latency period seen before proliferation is effected, maybe due a number of potential mechanisms. The simplest explanation wouldbe the accumulation with time of some active element or elements. Insuch a case one would expect the effect to increase in time with somelinear or exponential kinetics. However, the kinetics seem to be Sshaped, i.e. initially (for 12 hours) having no, or very little effect,and then picking up momentum and reaching a steady-state at about 24hours. The initial segment of such behavior is typical of cooperative ormulti-target processes. It is not likely that a 12 hour long delayfollowed by constant kinetics within an additional period of 12 hours becaused by diffusion processes. Such a behavior may however be theproduct of a link to the typical 24 hour division cycle of the celllines involved. Thus, if the TTFields effect on proliferation requiresthe disruption of two processes (“two hits”) that occur at two differentpoints in time that are 12 hours apart in the division cycle, theobserved behavior would result. Within this framework the roughly 12hour delay is the result of the difference in time between the two “hitpoints” in the cycle. Since the division of the numerous cells involvedis not synchronous, it would take an additional period of 12 hours toaffect all the dividing cells.

It may be that the unique combined effect of TTFields withCyclophosphamide results from the fact that the chemical agent, which ispresent throughout the studied period, disrupts one of the two “hitpoints” or targets, thus rendering the TTFields effect a regular “singlehit” one. The above is consistent, for example, with one target beingpart of G₁ while the other is part of G₂. However, there is anindication that TTFields effect the spindle microtubulepolymerization—depolymerization stage and Cyclophosphamide effects the Sphase. Therefore it is reasonable to assume that one TTFields target isin G₂ while the other is part of the S phase where Cyclophosphamide canreplace its action.

The results of the experiments reported above support the notion thatTTFields may be used as an effective adjunct to enhance the effects ofcurrently used chemotherapeutic agents. This may provide an idealcombination having additive to synergistic efficacy and potentiallywithout an increase in toxicity. Moreover, as seen in FIG. 8 forCyclophosphamide, the combination with TTFields produces the sametherapeutic effect using concentrations of 1 mM, as compared with 30 mMusing the drug alone. This dose reduction will most likely result insignificantly lower drug side effects. An additional potential benefitis the outcome of the fact that TTFields are physical agent the actionof which does not depend on specific cell receptors and thus may beeffective over a broad range of malignancies. This wide range efficacyis similar to that of irradiation, but without the severe side effectsassociated with irradiation. The predicted potential benefits are basedon the fact that in pilot clinical trials long term treatment withTTFields was not associated with any significant adverse side effects.

In addition to the five particular drugs discussed above, TTFields canbe used in conjunction with other anti-cancer treatments. Examples ofother anti-cancer treatments that can be combined with TTFields include,but are not limited to, five general categories:

The first categories is surgery, including but not limited to opensurgery, laparoscopic surgery, minimal resection surgery, debulkingsurgery, complete resection surgery, etc. The second category is localablation techniques including but not limited to radio-surgery, RFablation, and focused ultrasound. For these first two categories, theTTFields may be applied before the surgery or ablation to shrink thetumor, and/or after the surgery or ablation to deal with any remainsthereof The third category is ionizing radiation using various dosingand focusing regimen including but not limited to whole organ radiation(e.g., brain), regional radiation (e.g. Y shaped), focal radiation,single dose radiation, fractionated dose radiation, andhyper-fractionated dose radiation.

The fourth category is chemotherapy, including but not limited to {a}Alkylating agents that act mainly by forming covalent bonds between DNAbases, including but not limited to Nitrogen Mustards (e.g.,Cyclophosphamide), Aziridines and Epoxides (e.g., Thiopeta), AlkylSulfonates (e.g. Busulfan), Nitrosureas (e.g., BCNU and CCNU), Hydrazineand Triazine derivatives (e.g., Procarbazine and Temozolomide); {b}Cisplatin and its analogs that act by forming DNA adducts which lead tointra-strand and inter-strand linking leading to the formation of DNAfilaments, including but not limited to Carboplatin, Cisplatin, andOxaliplatin; {c} Antimetabolites including but not limited to Folatemetabolism inhibitors (e.g., Methotrexate, Trimetrexate, Tomudex),5-fluoropyrimidines (e.g., 5-FU), Oral Fluoropyramidines (e.g., Tegafur,Uracil, Capecitabine), Necleoside analogs (e.g., Cytarabine),Gemcitabine, and 6-thiopurines (e.g., 6-MP and 6-TG); {d} TopoisomeraseInteractive Agents that affect the topologic states of DNA byinterfering or modulating DNA cleavage, strand passage and re-ligation,including but not limited to Epipodophyllotoxins (e.g., Etoposide andTeniposide), Camptothecin Analogs, Anthracyclines (e.g., Doxorubicin,Daunorubicin, Epirubicin, Idarubicin), Mitoxantrone and Losoxantrone,and Dactinomycin; {e} Antimicrotubule Agents, which interfere with theproper polymerization/depolymerization of microtubules, including butnot limited to Vinca alkaloids (e.g., Vincristine, Vinorelbine andVinblastine), Taxanes (e.g., Paclitaxel, Docetaxel), and EstramustinePhosphate; and {f} Numerous miscellaneous agents exist which cannot beclassified into any of the above groups, including but not limited toSuramin, Bleomycin, L-Asparaginase, and Amifostine.

The fifth category is biological therapies, including but not limited to{a} Inteferons; {b} Interleukin-2; {c} Hormonal therapies including butnot limited to Tamoxifen, Toremifene, Raloxifene, Medroxyprogesteroneand Megestrol, Aromatase inhibitors, GNRH analogues, Antiandrogens,Diethylstilbesterol and Estradiol, and Octreotide; {d} Differentiationagents that catalyze the differentiation of cancerous cells into theirmature (differentiated) forms and then to programmed cell death,including but not limited to Retinoids (e.g., All-Trans-Retinoic Acid),Arsenic Trioxide, Histone Deacetylase inhibitors, Vitamin D, andCytokines; {e} Therapeutic Monoclonal Antibodies; and {f}Antiangiogenesis agents (e.g., VEGF inhibitors).

In addition to the in vitro data discussed above, preliminaryexperiments on live animals with VX2 tumors treated using a combinationof Doxil and TTFields show a significant reduction in tumor growth ratefor combination therapy as compared to treatment using Doxil alone orTTFields alone. Since TTFields show no systemic toxicities, it appearsthat TTFields can be applied to patients before, during and/or after anyother anti-cancer treatment to combat the cancer using two differentmodalities. The dosages, strengths, and timing of the various treatmentsmay be changed to optimize the results that are desired. Note that themost beneficial combination regimen may differ considerably depending onthe type of cancer treated, the exact stage of the disease and the typeof anticancer treatment used, it should be relatively simple todetermine the best combination regimen experimentally. TTFields can alsobe applied together with more than one of the other anti-cancerapproaches.

FIG. 12 is an example of an apparatus that is suitable for use intreating live patients with combined TTField and drug therapy, and itmay be used in combination with any conventional drug delivery mechanism(not shown) to implement the combined TTField and drug therapy. FIG. 12is a simple schematic diagram of the electronic apparatus 200illustrating the major components thereof The electronic apparatus 200generates the desired electric waveforms. The apparatus 200 includes agenerator 210 and a pair of conductive leads 220 that are attached atone end thereof to the generator 210. The opposite ends of the leads 220are connected to insulated conductors 230 that are activated by theelectric signals (e.g., waveforms). The insulated conductors 230 arealso referred to hereinafter as isolects 230. Optionally and accordingto another exemplary embodiment, the apparatus 200 includes atemperature sensor 240 and a control box 250 which are both added tocontrol the amplitude of the electric field generated so as not togenerate excessive heating in the area that is treated.

The generator 210 generates an alternating voltage waveform atfrequencies in the range from about 50 KHz to about 500 KHz (preferablyfrom about 100 KHz to about 300 KHz). The required voltages are suchthat the electric field intensity in the tissue to be treated is in therange of about 0.1 V/cm to about 10 V/cm, and preferable between about 1V/cm and about 5 V/Cm. To achieve this field, the actual potentialdifference between the two conductors in the isolects 230 is determinedby the relative impedances of the system components, as described below.

When the control box 250 is included, it controls the output of thegenerator 210 so that it will remain constant at the value preset by theuser or the control box 250 sets the output at the maximal value thatdoes not cause excessive heating, or the control box 250 issues awarning or the like when the temperature (sensed by temperature sensor240) exceeds a preset limit.

The leads 220 are standard isolated conductors with a flexible metalshield, preferably grounded so that it prevents the spread of theelectric field generated by the leads 220. The isolects 230 havespecific shapes and positioning so as to generate an electric field ofthe desired configuration, direction and intensity at the target volumeand only there so as to focus the treatment.

The specifications of the apparatus 200 as a whole and its individualcomponents are largely influenced by the fact that at the frequency ofthe TTFields (50 KHz-500 KHz), living systems behave according to their“Ohmic”, rather than their dielectric properties. The only elements inthe apparatus 200 that behave differently are the insulators of theisolects 230 (see FIGS. 14-15). The isolects 200 consist of a conductorin contact with a dielectric that is in contact with the conductivetissue thus forming a capacitor.

The details of the construction of the isolects 230 is based on theirelectric behavior that can be understood from their simplified electriccircuit when in contact with tissue as generally illustrated in FIG. 13.In the illustrated arrangement, the potential drop or the electric fielddistribution between the different components is determined by theirrelative electric impedance, i.e., the fraction of the field on eachcomponent is given by the value of its impedance divided by the totalcircuit impedance. For example, the potential drop on elementΔV_(A)=A/(A+B+C+D+E). Thus, for DC or low frequency AC, practically allthe potential drop is on the capacitor (that acts as an insulator). Forrelatively very high frequencies, the capacitor practically is a shortand therefore, practically all the field is distributed in the tissues.At the frequencies of the TTFields (e.g., 50 KHz to 500 KHz), which areintermediate frequencies, the impedance of the capacitance of thecapacitors is dominant and determines the field distribution. Therefore,in order to increase the effective voltage drop across the tissues(field intensity), the impedance of the capacitors is to be decreased(i.e., increase their capacitance). This can be achieved by increasingthe effective area of the “plates” of the capacitor, decrease thethickness of the dielectric or use a dielectric with high dielectricconstant.

In order to optimize the field distribution, the isolects 230 areconfigured differently depending upon the application in which theisolects 230 are to be used. There are two principle modes for applyingthe TTFields. First, the TTFields can be applied by external isolectsand second, the TTFields can be applied by internal isolects.

TTFields that are applied by external isolects can be of a local type orwidely distributed type. The first type includes, for example, thetreatment of skin tumors and treatment of lesions close to the skinsurface. FIG. 14 illustrates an exemplary embodiment where the isolects230 are incorporated in a skin patch 300. The skin patch 300 can be aself-adhesive flexible patch with one or more pairs of isolects 230. Thepatch 300 includes internal insulation 310 (formed of a dielectricmaterial) and the external insulation 260 and is applied to skin surface301 that contains a tumor 303 either on the skin surface 301 or slightlybelow the skin surface 301. Tissue is generally indicated at 305. Toprevent the potential drop across the internal insulation 310 todominate the system, the internal insulation 310 must have a relativelyhigh capacity. This can be achieved by a large surface area; however,this may not be desired as it will result in the spread of the fieldover a large area (e.g., an area larger than required to treat thetumor). Alternatively, the internal insulation 310 can be made very thinand/or the internal insulation 310 can be of a high dielectric constant.As the skin resistance between the electrodes (labeled as A and E inFIG. 13) is normally significantly higher than that of the tissue(labeled as C in FIG. 13) underneath it (1-10 KΩ vs. 0.1-1 KΩ), most ofthe potential drop beyond the isolects occurs there. To accommodate forthese impedances (Z), the characteristics of the internal insulation 310(labeled as B and D in FIG. 13) should be such that they have impedancepreferably under 100 KΩ at the frequencies of the TTFields (e.g., 50 KHzto 500 KHz). For example, if it is desired for the impedance to be about10 K Ohms or less, such that over 1% of the applied voltage falls on thetissues, for isolects with a surface area of 10 mm², at frequencies of200 KHz, the capacity should be on the order of 10⁻¹⁰ F., which meansthat using standard insulations with a dielectric constant of 2-3, thethickness of the insulating layer 310 should be about 50-100 microns. Aninternal field 10 times stronger would be obtained with insulators witha dielectric constant of about 20-50.

Using an insulating material with a high dielectric constant increasesthe capacitance of the electrodes, which results in a reduction of theelectrodes' impedance to the AC signal that is applied by the generator1 (shown in FIG. 12). Because the electrodes A, E are wired in serieswith the target tissue C, as shown in FIG. 13, this reduction inimpedance reduces the voltage drop in the electrodes, so that a largerportion of the applied AC voltage appears across the tissue C. Since alarger portion of the voltage appears across the tissue, the voltagethat is being applied by the generator 1 can be advantageously loweredfor a given field strength in the tissue.

The desired field strength in the tissue being treated is preferablybetween about 0.1 V/cm and about 10 V/cm, and more preferably betweenabout 2 V/cm and 3 V/cm or between about 1 V/cm and about 5 V/cm. If thedielectric constant used in the electrode is sufficiently high, theimpedance of the electrodes A, E drops down to the same order ofmagnitude as the series combination of the skin and tissue B, C, D. Oneexample of a suitable material with an extremely high dielectricconstant is CaCu₃Ti₄O₁₂, which has a dielectric constant of about 11,000(measured at 100 kHz). When the dielectric constant is this high, usefulfields can be obtained using a generator voltage that is on the order ofa few tens of Volts.

Since the thin insulating layer can be very vulnerable, etc., theinsulation can be replaced by very high dielectric constant insulatingmaterials, such as titanium dioxide (e.g., rutile), the dielectricconstant can reach values of about 200. There a number of differentmaterials that are suitable for use in the intended application and havehigh dielectric constants. For example, some materials include: lithiumniobate (LiNbO₃), which is a ferroelectric crystal and has a number ofapplications in optical, pyroelectric and piezoelectric devices; yttriumiron garnet (YIG) is a ferromagnetic crystal and magneto-opticaldevices, e.g., optical isolator can be realized from this material;barium titanate (BaTiO₃) is a ferromagnetic crystal with a largeelectro-optic effect; potassium tantalate (KTaO₃) which is a dielectriccrystal (ferroelectric at low temperature) and has very low microwaveloss and tunability of dielectric constant at low temperature; andlithium tantalate (LiTaO₃) which is a ferroelectric crystal with similarproperties as lithium niobate and has utility in electro-optical,pyroelectric and piezoelectric devices. Insulator ceramics with highdielectric constants may also be used, such as a ceramic made of acombination of Lead Magnesium Niobate and Lead Titanate. It will beunderstood that the aforementioned exemplary materials can be used incombination with the present device where it is desired to use amaterial having a high dielectric constant.

One must also consider another factor that affects the effectivecapacity of the isolects 230, namely the presence of air between theisolects 230 and the skin. Such presence, which is not easy to prevent,introduces a layer of an insulator with a dielectric constant of 1.0, afactor that significantly lowers the effective capacity of the isolects230 and neutralizes the advantages of the titanium dioxide (rutile),etc. To overcome this problem, the isolects 230 can be shaped so as toconform with the body structure and/or (2) an intervening filler 270 (asillustrated in FIG. 16C), such as a gel, that has high conductance and ahigh effective dielectric constant, can be added to the structure. Theshaping can be pre-structured (see FIG. 16A) or the system can be madesufficiently flexible so that shaping of the isolects 230 is readilyachievable. The gel can be contained in place by having an elevated rimas depicted in FIGS. 16C and 16C′. The gel can be made of hydrogels,gelatins, agar, etc., and can have salts dissolved in it to increase itsconductivity. FIGS. 16A-16C′ illustrate various exemplary configurationsfor the isolects 230. The exact thickness of the gel is not important solong as it is of sufficient thickness that the gel layer does not dryout during the treatment. In one exemplary embodiment, the thickness ofthe gel is about 0.5 mm to about 2 mm. Preferably, the gel has highconductivity, is tacky, and is biocompatible for extended periods oftime. One suitable gel is AG603 Hydrogel, which is available from AmGelTechnologies, 1667 S. Mission Road, Fallbrook, Calif. 92028-4115, USA.

In order to achieve the desirable features of the isolects 230, thedielectric coating of each should be very thin, for example from between1-50 microns. Since the coating is so thin, the isolects 230 can easilybe damaged mechanically or undergo dielectric breakdown. This problemcan be overcome by adding a protective feature to the isolect'sstructure so as to provide desired protection from such damage. Examplesof some suitable protective features are described in publishedapplication US2005/0209642, which is incorporated herein by reference.

However, the capacity is not the only factor to be considered. Thefollowing two factors also influence how the isolects 230 areconstructed. The dielectric strength of the internal insulating layer310 and the dielectric losses that occur when it is subjected to theTTFields, i.e., the amount of heat generated. The dielectric strength ofthe internal insulation 310 determines at what field intensity theinsulation will be “shorted” and cease to act as an intact insulation.Typically, insulators, such as plastics, have dielectric strength valuesof about 100V per micron or more. As a high dielectric constant reducesthe field within the internal insulator 310, a combination of a highdielectric constant and a high dielectric strength gives a significantadvantage. This can be achieved by using a single material that has thedesired properties or it can be achieved by a double layer with thecorrect parameters and thickness. In addition, to further decreasing thepossibility that the insulating layer 310 will fail, all sharp edges ofthe insulating layer 310 should be eliminated as by rounding thecorners, etc., as illustrated in FIG. 16D using conventional techniques.

FIG. 15 illustrates a second type of treatment using the isolects 230,namely electric field generation by internal isolects 230. A body towhich the isolects 230 are implanted is generally indicated at 311 andincludes a skin surface 313 and a tumor 315. In this embodiment, theisolects 230 can have the shape of plates, wires or other shapes thatcan be inserted subcutaneously or a deeper location within the body 311so as to generate an appropriate field at the target area (tumor 315).

It will also be appreciated that the mode of isolects application is notrestricted to the above descriptions. In the case of tumors in internalorgans, for example, liver, lung, etc., the distance between each memberof the pair of isolects 230 can be large. The pairs can even bypositioned opposite sides of a torso 410, as illustrated in FIG. 17. Thearrangement of the isolects 230 in FIG. 17 is particularly useful fortreating a tumor 415 associated with lung cancer or gastro-intestinaltumors. In this embodiment, the TTFields spread in a wide fraction ofthe body. Note also that in addition to external electrode embodimentsdescribed above, the combined TTField and drug treatment may beimplemented using the internal probe embodiments described in publishedapplication US2005/0209642, which is incorporated herein by reference.

In order to avoid overheating of the treated tissues, a selection ofmaterials and field parameters is needed. The isolects insulatingmaterial should have minimal dielectric losses at the frequency rangesto be used during the treatment process. This factor can be taken intoconsideration when choosing the particular frequencies for thetreatment. The direct heating of the tissues will most likely bedominated by the heating due to current flow (given by the I*R product).In addition, the isolect (insulated electrode) 230 and its surroundingsshould be made of materials that facilitate heat losses and its generalstructure should also facilitate head losses, i.e., minimal structuresthat block heat dissipation to the surroundings (air) as well as highheat conductivity. Using larger electrodes also minimizes the localsensation of heating, since it spreads the energy that is beingtransferred into the patient over a larger surface area. Preferably, theheating is minimized to the point where the patient's skin temperaturenever exceeds about 39° C.

Another way to reduce heating is to apply the field to the tissue beingtreated intermittently, by applying a field with a duty cycle betweenabout 20% and about 50% instead of using a continuous field. Forexample, to achieve a duty cycle of 33%, the field would be repetitivelyswitched on for one second, then switched off for two seconds.Preliminary experiments have shown that the efficacy of treatment usinga field with a 33% duty cycle is roughly the same as for a field with aduty cycle of 100%. In alternative embodiments, the field could beswitched on for one hour then switched off for one hour to achieve aduty cycle of 50%. Of course, switching at a rate of once per hour wouldnot help minimize short-term heating. On the other hand, it couldprovide the patient with a welcome break from treatment.

It will also be appreciated that the present apparatus can furtherinclude a device for rotating the TTFields relative to the livingtissue. For example and according to one embodiment, the alternatingelectric potential applies to the tissue being treated is rotatedrelative to the tissue using conventional devices, such as a mechanicaldevice that upon activation, rotates various components of the presentsystem.

The TTFields may be applied to different pairs of the insulatedelectrodes 230 in a consecutive manner in order to vary the direction ofthe TTFields that travel through the target region, as described inpublished application US2005/0209642, which is incorporated herein byreference. The changing of the field's direction may be implemented in astepwise manner or in a continuous manner, also as described inpublished application US2005/0209642.

As described in published application US2005/0209642, it can beadvantageous to apply a distribution of different frequencies to thepopulation. For example, experiments indicate that using two frequenciesof 170 kHz and 250 kHz to destroy a population of glioma cells would bemore effective than using a single frequency of 200 kHz. When more thanone frequency is used, the various frequencies may be appliedsequentially in time. For example, in the case of glioma, fieldfrequencies of 100, 150, 170, 200, 250, and 300 kHz may be appliedduring the first, second, third, fourth, fifth, and sixth minutes oftreatment, respectively. That cycle of frequencies would then repeatduring each successive six minutes of treatment. Alternatively, thefrequency of the field may be swept in a stepless manner from 100 to 300kHz. Optionally, this frequency cycling may be combined with thedirectional changes described above.

In an alternative embodiment, a signal that contains two or morefrequencies components simultaneously (e.g., 170 kHz and 250 kHz) isapplied to the electrodes to treat a populations of cells that have adistribution of sizes. The various signals will add by superposition tocreate a field that includes all of the applied frequency components.

As used herein, the term “tumor” refers to a malignant tissue comprisingtransformed cells that grow uncontrollably. In addition, the presentinvention can control uncontrolled growth associated with non-malignantor pre-malignant conditions, and other disorders involving inappropriatecell or tissue growth by application of an electric field in accordancewith the invention to the tissue undergoing inappropriate growth.

Furthermore, undesirable fibroblast and endothelial cell proliferationassociated with wound healing, leading to scar and keloid formationafter surgery or injury, and restenosis after angioplasty or placementof coronary stents can be inhibited by application of an electric fieldin accordance with the present invention. The non-invasive nature ofthis invention makes it particularly desirable for these types ofconditions, particularly to prevent development of internal scars andadhesions, or to inhibit restenosis of coronary, carotid, and otherimportant arteries.

Thus, the present invention provides an effective, simple method ofselectively destroying dividing cells, e.g., tumor cells and parasiticorganisms, while non-dividing cells or organisms are left affected byapplication of the method on living tissue containing both types ofcells or organisms. Thus, unlike many of the conventional methods, thepresent invention does not damage the normal cells or organisms. Inaddition, the present invention does not discriminate based upon celltype (e.g., cells having differing sizes) and therefore may be used totreat any number of types of sizes having a wide spectrum ofcharacteristics, including varying dimensions.

While the invention has been particularly shown and described withreference to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details can bemade without departing from the spirit and scope of the invention.

1. A method of killing or inhibiting the growth of cancer cells in atarget region, the method comprising the steps of: applying, to thetarget region, an AC electric field that damages the cancer cells orinhibits the growth of the cancer cells, but leaves normal cells in thetarget region substantially unharmed, wherein the electric field has afrequency between 50 kHz and 500 kHz and wherein the direction of thefield is repeatedly changed during a course of treatment by applying thefield between different sets of electrodes; and treating the cancercells with an other anti-cancer regimen, wherein the applying step andthe treating step are performed simultaneously.
 2. The method of claim1, wherein the other anti-cancer regimen comprises treating the cancercells with an anti-cancer drug.
 3. A method of inhibiting the growth ofcancer cells in a target region, the method comprising the steps of:treating the cancer cells with an anti-cancer drug; and applying an ACelectric field to the target region for a period of time, wherein theelectric field has frequency and field strength characteristics selectedto inhibit the growth of cancer cells in the target region and whereinthe electric field has a frequency between 50 kHz and 500 kHz andwherein the direction of the field is repeatedly changed during a courseof treatment by applying the field between different sets of electrodes.4. A method of killing or inhibiting the growth of cancer cells in atarget region, the method comprising the step of Applying, to the targetregion, an AC electric field that damages the cancer cells or inhibitsthe growth of the cancer cells, but leaves normal cells in the targetregion substantially unharmed, wherein the AC electric field is appliedwhile the cancer cells are being treated with an anti-cancer drug,wherein the electric field has frequency between about 100 kHz and 300kHz, and a field strength in the target region of at least 1 V/cm andwherein the direction of the field is repeatedly changed during a courseof treatment by applying the field between different sets of electrodes.5. The method of claim 4, wherein the drug comprises Cyclophosphamide.6. The method of claim 4, wherein the period of time is at least 6hours.
 7. The method of claim 6, wherein the frequency is about 150 kHz.8. The method of claim 6, wherein the frequency is about 200 kHz.
 9. Themethod of claim 4, wherein the drug dosage is less than 20% of astandard dosage for the drug.
 10. The method of claim 4, wherein theperiod of time is at least 24 hours.
 11. The method of claim 10, whereinthe drug comprises at least one of Paclitaxel, Doxorubicin,Cyclophosphamide, and Cisplatin.
 12. The method of claim 4, wherein thefield strength is between 1 V/cm and 5 V/cm and the period of time is atleast 24 hours.
 13. The method of claim 12, wherein the frequency isabout 150 kHz.
 14. The method of claim 12, wherein the frequency isabout 200 kHz.